Oxygen is essential for living organisms, but it also has a dangerous aspect of causing unwanted oxidation of organisms' components. Living organisms have a series of defense mechanisms against such an oxidative stress. Vitamin E is one of important anti-oxidative substances in the living organisms. Among organism's components, a biomembrane phospholipid (particularly, a polyunsaturated fatty acid chain in phospholipid) is most likely to receive an attack by oxygen. When biomembranes undergo an oxidative impairment, the membrane permeability increases, and finally leading to cell death. Vitamin E is lipid-soluble (lipophilic) and usually buried in biomembrane bilayers in cells. It plays the most dominant role in preventing biomembrane oxidation.
As a protein cable of specifically binding to this vitamin E, α-TTP was isolated from a soluble fraction of rat liver and found to enhance intermembrane transfer (Eur. J. Biochem. 177, 537, 1981). In addition, purification and gene structure analysis were also performed on this protein (J. Biol. Chem. 268, 17705, 1993; Biochem. J. 306, 437, 1995). Further, human α-TTP gene was shown to be a causative gene per se for a hereditary disease called “familial isolated vitamin E deficiency (FIVE deficiency)” (Nature Genetics 9, 141, 1995).
Familial isolated vitamin E deficiency has previously been known as a hereditary disease which produces no increase in vitamin E level in the body, even when much vitamin E is taken into the body. A patient with this disease suffers from necrosis of nerves, particularly sensory nerves, and in a serious case he will die around 20 years of age. Vitamin E circulates in the blood as a conjugate with plasma lipoprotein and then enters peripheral tissues. Meanwhile, plasma lipoprotein is secreted from the liver and finally returns to the liver to be metabolized. Vitamin E absorbed from food through blood vessels is incorporated onto lipoprotein (VLDL) in the liver. In this hereditary disease, an impairment is found in just this process, i.e., an incorporation process of vitamin E onto VLDL in the liver.
In view of these circumstances, the object of the present invention is to provide a non-human mammal useful in analyzing α-TTP functions and developing a therapeutic agent for diseases caused by α-TTP mutation. In the prior art, an animal could enter a vitamin E deficient state by feeding it with a vitamin E-deficient diet, but in a normal animal, a very long period of time was required to achieve complete clearance of vitamin E from the animal body because the normal animal has an efficient re-circulation system for vitamin E (this system involves α-TTP). In the present invention, an animal with a disrupted α-TTP gene involved in this vitamin E re-circulation system is created to provide a congenitally vitamin E-deficient animal model highly sensitive to oxidative stress, which is advantageous in understanding diseases caused by vitamin E deficiency, i.e., deficiency of anti-oxidative substances critical for the body, and in developing a therapeutic agent for such diseases. More specifically, the object of the present invention is to provide a knockout animal artificially modified to inhibit α-TTP gene expression and a method for producing the animal.